Prevalence of Cervical Dysplasia in HIV-Positive and HIV-Negative Women at the Sihanouk Hospital Center of HOPE, Phnom Penh , Cambodia

Introduction: There is a high burden of cervical cancer in Cambodia, yet published data on the prevalence of cervical dysplasia and the risk factors contributing to the development of pre-cancerous lesions in Cambodian women is very limited. In addition, as it is well known that HIV positivity increases cervical cancer risk, it is important to quantify the prevalence of cervical dysplasia and carcinoma among Cambodian women living with HIV disease. Methods: A cross-sectional study was conducted with a sample of 499 HIV+ and 501 HIV- Cambodian women at the Sihanouk Hospital Center of HOPE. Visual inspection with 5% acetic acid was the method of screening. Colposcopy was performed on all VIA+ patients, and subsequent treatment followed WHO guidelines. Logistic regression models, stratified by both HIV+ and HIV- groups, were used to assess significant factors associated with having dysplasia. Results: VIA+ results were prevalent in both the HIV+ and HIV- arms of the study. The HIV+ patients were more likely to have a lower age at coitarche, lower weight, 2 or more lifetime sexual partners, two or greater pregnancies, or be unmarried. The estimated prevalence of VIA detected cervical dysplasia was 11% for the entire study sample, 13.4% in the HIV positive (HIV+) group and 8.6% in the HIV negative (HIV-) group (OR: 1.65; 95% CI: 1.10, 2.48; p=0.01). For the HIV+ group, having a history of 4 or more full-term pregnancies (OR: 3.42; 95% CI: 1.01-11.64; p=0.049) was found to be significantly associated with having an increased risk of developing cervical dysplasia in the multivariate model. Conclusion: Cervical dysplasia is prevalent in both HIV positive and negative Cambodian women and a VIA based national screening programs need to be developed and expanded to provide access to affordable and effective treatment for cervical dysplasia and cancers.

Introduction associated with having cervical dysplasia in Cambodian women is not known.
In recent years, Cambodia has experienced a dramatic decline in its HIV infection rates and the prevalence of HIV in Cambodia, is relatively low (Vun et al., 2014;Pe et al., 2015). However, there are still approximately 39,000 females, 15 years and older, living with HIV in Cambodia. In addition, a cervical cancer diagnosis is considered to be AIDS defining in a HIV+ woman. Furthermore, the risk of developing cervical dysplasia and invasive cervical cancer is significantly higher in women living with HIV disease and the rate of progression of precancerous cervical lesions to invasive disease is faster in this high-risk group compared to HIV-females (Maiman et al., 1997;Orem et al., 2004;Massad et al., 2015).
As logistical and economic issues make traditional cytology-based cervical cancer screening challenging in resource-limited countries such as Cambodia, alternative screening strategies have been developed. One such strategy, visual inspection with acetic acid (VIA) is suitable for low-resource settings because it is inexpensive and does not require difficult-to-obtain materials (Lim et al., 2011;Sahasrabuddhe et al., 2012). VIA involves direct visualization of the cervix for the presence of pre-cancerous cervical lesion after the application of acetic acid 3-5% (Sankaranarayanan et al., 2004). As the results of the VIA are immediate, a women's health specialist may treat most pre-cancerous lesions with cryotherapy in the same visit. This "screen-and-treat" strategy has been shown to be relatively easy to implement and very effective in reducing cervical cancer in other low-resource settings (Nuranna et al., 2012;Ramogola-Masire et al., 2012).
As previously mention, there are very limited data regarding prevalence of cervical dysplasia in Cambodia. As such, the primary aim of this study is to determine the prevalence of VIA-detected cervical dysplasia in women with, and without, HIV disease. The availability of these data would inform the allocation of resources and aid in the development of relevant national programs, such as effective screen-and-treat programs, to adequately address cervical dysplasia and carcinoma in Cambodia. Additionally, this study was designed to see if any additional risk factors might increase the risk of developing cervical dysplasia in Cambodian women so that limited resources may be focused on the most at risk women.

Study Location and Duration
This study was conducted from October 2014 to September 2015 at the Sihanouk Hospital Center of Hope (SHCH) in Phnom Penh, Cambodia and was approved by the National Ethics Committee for Health Research in Cambodia. SHCH was founded in 1996 with a dual mission to train medical professionals and provide healthcare to the poor and vulnerable populations in the country. Despite being located in the Cambodian capital, due to its extensive outreach programs, SCHC services women from both rural and urban areas throughout Cambodia. As such, the convenience sample of study participants used in this study represents a cross-section of Cambodian women.

Study Population
The study population was one thousand 30-49 yearold, non-pregnant Cambodian women. Study enrollment was stratified by the participant's HIV status at the time of enrollment. If, at enrollment, a given participant's HIV status was unknown, a rapid diagnostic test was conducted to determine their HIV status. Following enrollment, a physician-administered questionnaire captured demographic, behavioral and biological characteristics and information including parity, age, age at coitarche, lifetime number of sexual partners, hormonal contraceptive use, history of smoking, current marital status and cervical cancer screening history. For HIV+ participants, data on CD4 cell count, use of highly active antiretroviral therapy (HAART) and number of months on HAART was obtained from available patient medical records.

VIA Screening and Treatment
The lead physician (ST) assisted by another physician or a nurse conducted the VIA screening for this study. VIA was performed and interpreted based on the recommendations of the World Health Organization (WHO, 2013) the International Agency on Cancer Research and the Cambodian Ministry of Health in its standard operating procedures manual. During the same clinic visit, and after VIA screening had been performed, VIA+ participants were treated with cryotherapy. For participants who tested VIA+ and who had cervical lesions ineligible for treatments with cryotherapy (based on WHO guidelines), or who a suspected invasive cervical cancer, colposcopy and biopsy were performed at the clinic visit to aid in further evaluation and diagnosis of the observed cervical lesions. Based on the biopsy results, treatment in the form of a loop electrosurgical excision procedure (LEEP) for cervical intraepithelial neoplasia 2 or 3 (CIN2/ CIN3) or radical hysterectomy in more advanced cases was subsequently performed. Collected data were entered into a secure electronic database (EpiData) by trained data entry personnel. As part of quality control of the stored data, a random 10% of the study sample was independently examined for accuracy and completeness.

Statistical Analysis
Descriptive statistics including means with associated standard deviations (SD) for continuous variables and proportions for categorical variables were calculated to describe the characteristics of the study participants. Estimation of descriptors was done first for the entire study sample and then by HIV status for the two subgroups. Differences in baseline characteristics between the HIV+ and HIV-subgroups were evaluated using t-test for continuous and chi-square test for categorical variables.
The prevalence of all VIA detected cervical dysplasia was calculated for the entire study sample and by the participants' HIV status. To determine the significant demographic, behavioral and biological factors associated with having a cervical dysplasia/carcinoma, univariate and Prevalence of Cervical Dysplasia in Phnom Penh, Cambodia mean age of participants was 39 years (SD±5.78), and the mean age at coitarche was 21.58 years (SD±4.57). In the study sample, 54.7% of participants had never received cervical cancer screening, and 11.2% reported the use of hormonal contraceptives (Table 1). Compared to the HIV-group, the HIV+ group had a significantly lower age at coitarche, lower weight, were more likely to report having 2 or more lifetime sexual partners, were less likely to be married, and more likely to have had two or greater pregnancies (Table 1). The prevalence of VIA detected cervical dysplasia among the total study participants was 11% (Table 2). multivariate logistic regression models were run using covariates listed in Table 1. Risk factors included in the final multivariate model were selected a priori and this final multivariate model was run using a p-value of 0.05. All statistical analyses of this study were conducted using SAS 9.4 (SAS Institute, Inc).

Participant Characteristics
A total of 1,000 participants were recruited into the study, of which 499 were HIV+ and 501 were HIV-. The

Predictors of VIA Detected Cervical Dysplasia
Unadjusted and adjusted regression models were run for the entire cohort of 1,000 participants (

Discussion
Our findings indicate a significant prevalence of VIA detected cervical dysplasia in both HIV positive (13.4%) and HIV negative (8.6%) women in Cambodia. The prevalence of high grade dysplasia was 0.8% for HIV negative women and 1.0% for HIV positive women. In comparison, Hav et al., (2016) previously showed a 6.25% cervical dysplasia risk in HIV-negative women detected by cytology-based screening. They acknowledge, however, that a high rate of unsatisfactory pap smears in their study may have falsely lowered the detection rate of dysplasia. Rageunaud et al., (2009) reported on an exploratory cytology-based screening program of 200 HIV+ Cambodian women. This study, conducted in 2007, reported that 17% of their patients had biopsy proven cervical dysplasia/carcinoma. They also reported a high loss to follow up rate in their study and they recommended that a VIA based screening-and-treat strategy be explored for all HIV+ Cambodian women. Lastly, Lim et al reported on a study comparing VIA versus cytology based screening in 304 HIV+ Cambodian women (Lim et al., 2011). They reported that 19% of women (n=55) screened positive with VIA, however, cytology was negative for dysplasia in 67% (n=37) of these VIA+ women . Unfortunately, resource limitations prevented the authors from obtaining cervical biopsies to provide histologic confirmation of dysplasia. As such, they acknowledge that they were unable to determine the accuracy of VIA as compared to Pap smear in HIV infected women.
All the risk factors we examined (age, smoking, number of partners, age at sexual debut, number of full term pregnancies, weight, marital status, previous cervical cancer screening, use of hormonal contraceptives, and family history) have been shown by previous studies to have a significant association with cervical abnormalities (International Collaboration of Epidemiological Studies of Cervical, 2007;Chelimo et al., 2013;Vaccarella et al., 2013). However, only family history was found to be significantly associated with the odds of having a cervical dysplasia in our HIV-group, and the 95% CI were wide. The lack of association may be due to the generally low prevalence of many of these risk factors among Cambodian women in this study as well as in the whole country (CDHS, 2014). For example, in our study, only 0.8% of participants reported a history of smoking. Also in the case of weight, the reported prevalence of obesity in the female Cambodian population is less than 3% (27) and in our study the mean weight of participants was found to be only 53.77 kg (SD±8.68).
For the entire cohort and the HIV+ group, 4 or more full-time pregnancies were associated with -an increased risk of developing cervical dysplasia. Age at coitarche and number of partners were also associated in the univariate analysis of the entire cohort. Of course, it is likely that these risk factors are also associated with an increased risk of contracting high-risk human papilloma virus that is the causative factor in more than 99% of cervical cancer.
Limitations of this study include the fact that 44% of study participants had had previous cervical cancer screening and appropriate treatment. As such, the rate of dysplasia in our cohort may be significantly lower than in a cohort of women who have never received screening. Secondly, it might be argued that despite SHCH's extensive outreach into both urban and rural settings, that our convenience sample of women does not represent a true cross section of Cambodian women. Lastly, as only the VIA+ women who had lesions suspicious for high-grade dysplasia or carcinoma had confirmatory biopsies, it is possible that there were both false positives and negative VIAs.
In conclusion, the results of our study, in combination of the aforementioned other studies, support the establishment of a national VIA based screen-and treat screening program in Cambodia. In addition, while it is important that this service be offered to all Cambodian women we recommend that a screen and treat program should ideally be integrated into pre-existing HIV services. This will ensure that HIV positive women have easy access and are frequently screened per the WHO guidelines, to allow for early detection and management of abnormalities before they progress to an advance stage. While Cambodia's National Center for HIV/ AIDS, Dermatology and STDs (NCHADS) has not yet implemented a cervical screening program for WLWH, there is momentum building for increased focus on this population if funding allows.

List of Abbreviations
IARC -International Agency for Cancer Research VIA -Visual inspection with acetic acid SHCH -Sihanouk Hospital Center of Hope WLWH -Women living with HIV HAART -Highly active antiretroviral therapy CIN2/CIN3 -Cervical intraepithelial neoplasia 2/3

Trial Registration
This study was registered with clinicaltrials.gov on 17 September 2014. The trial registration number is NCT022537.

Ethics approval and consent
This study was approved by the National Ethics Committee for Health Research in Cambodia on 22 August 2014 (reference: 0251 NECHR). All participants gave voluntary, informed consent to participate.

Consent for publication
Not Applicable.

Availability of data and material
The datasets during and/or analysed during the current study available from the corresponding author on reasonable request.